Dengue virus encodes a polyprotein that is processed to mature gene products. The majority of the cleavages in the nonstructural (NS) protein region are effected by a virus-encoded protease, consisting of two proteins, NS2B and NS3. NS3 contains the protease active site, but the role of NS2B is less well understood. Recently, we showed that a forty amino acid domain in NS2B is required for protease activity. We have been pursuing two approaches to mutagenize dengue virus type 4 (DEN4) NS2B and NS3, in an effort to better understand this two component protease. Mutagenesis by selection. Previously, we cloned the DEN4 into the pKK223-3 vector under control of the tac promoter. This plasmid could be grown in E. coli JM110 in the absence of IPTG, but in E. coli HB101 only very tiny colonies would form. We reasoned that when the protease was expressed it was active and detrimental to E. coli. We selected rare large colonies on HB101, sequenced their plasmids, and found that they were all frameshift or nonsense mutations. To try to get around this problem, the lac Z' gene was cloned in-frame downstream of the protease. We transformed JM110 with this plasmid and selected large blue colonies in the presence of IPTG and XGAL. These should have been protease mutants, since they were large, and not frameshift or nonsense, since they were blue and thus expressed the lac Z' fragment. However, sequencing of several such putative mutant plasmids revealed mostly wild-type protease sequences, with two examples of nonsense mutations. We do not understand why the selection is not working as planned. Site-directed mutagenesis of NS2B. We have had made 20 oligos that can each be used to randomly mutagenize two consecutive residues of the forty amino acid essential domain of NS2B. Mutagenesis is being done in an M13 clone using a commercial kit. Mutants will then have to be subcloned into a pTM1 vector for analysis of their phenotypes. So far, we have had successful mutagenesis reactions with the first two oligos we have tried, and several mutants have been identified and are being subcloned.